CN103014174A - MPS (Macoplasmal Pneumoniae of Swine) PCR (Polymerase Chain Reaction) diagnostic kit - Google Patents
MPS (Macoplasmal Pneumoniae of Swine) PCR (Polymerase Chain Reaction) diagnostic kit Download PDFInfo
- Publication number
- CN103014174A CN103014174A CN2013100214754A CN201310021475A CN103014174A CN 103014174 A CN103014174 A CN 103014174A CN 2013100214754 A CN2013100214754 A CN 2013100214754A CN 201310021475 A CN201310021475 A CN 201310021475A CN 103014174 A CN103014174 A CN 103014174A
- Authority
- CN
- China
- Prior art keywords
- pcr
- diagnostic kit
- mps
- primer
- mycoplasmal pneumonia
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention discloses an MPS (Macoplasmal Pneumoniae of Swine) PCR (Polymerase Chain Reaction) diagnostic kit. The MPS PCR diagnostic kit comprises 0.1 moL/L of PBS (Phosphate Buffer Solution), a lysis buffer, DL2000, PCR enzyme, ultrapure water, primers, a positive contrast and a negative contrast, wherein the primers include an upstream primer and a downstream primer; the sequence of the upstream primer is 5'-TAGACCAGGATGGACAAGATGAT-3'; the sequence of the downstream primer is 5'-AACCACAGGACTAAGACGCAACA-3'. According to the invention, a pair of primers is designed based on the ompA gene sequence of the MPS of porcine actinobacillus pleuropneumonia in GenBank; and a PCR diagnostic kit for testing MPS is researched through optimization of PCR reaction conditions. The experimental result above shows that the MPS PCR diagnostic kit disclosed by the invention has the advantages of being fast, sensitive and accurate, and having good repeatability, long quality guarantee period and the like.
Description
Technical field
The present invention relates to a kind of test kit, especially a kind of porcine mycoplasmal pneumonia PCR diagnostic kit.
Background technology
Porcine mycoplasmal pneumonia (
Mycoplasma hyopneumoniae of swine, MPS) claim again mycoplasma pneumonia of swine, be by mycoplasma hyopneumoniae (
Mycoplasma hyopneumoniae, Mhp) cause a kind of high incidence of pig, the chronic respiratory transmissible disease of low actual (Straw B E etc., 1999).This disease is widely current in the world and exists, and its classical symptom is disease pig cough, expiratory dyspnea and asthma, and the major lesions feature is confluent bronchopneumonia, is " meat sample " or " shrimp sample " consolidation in sharp leaf, lobus cardiacus, middle leaf and the lobus diaphragmaticus leading edge of lung.This disease only betides pig, and the pig of different varieties, age and sex all can infect, and wherein with suckling pig and the susceptible of young pig, M ﹠ M is higher.Immunosuppression can occur behind the mycoplasma infection, and easy other transmissible diseases of secondary infection, thereby pig industry is caused comparatively serious financial loss (Yang Jiande etc., 2002).The detection of Mhp is normally based on separation or the immunofluorescence experiment of cause of disease.In recent years, for the separation of Mhp, serological identification and serum antibody monitoring had developed many methods, although these methods in theory can widespread use, but only have the good laboratory of minority condition just can accomplish, therefore, some non-specific methods remain useful.
Summary of the invention
The objective of the invention is: a kind of porcine mycoplasmal pneumonia PCR diagnostic kit is provided, and it can detect mycoplasma hyopneumoniae fast, and has special, sensitive, characteristics accurately, for the anti-system of porcine mycoplasmal pneumonia provides scientific basis.
The present invention is achieved in that porcine mycoplasmal pneumonia PCR diagnostic kit, and it comprises that concentration is the PBS40uL of 0.1moL/L, lysate 250uL, DL2000100uL, PCR enzyme 250uL, ultrapure water 170uL, primer 40uL, positive control 20uL and negative control 20uL; Wherein primer comprises upstream primer and downstream primer, each 20 uL of upstream primer and downstream primer, and the sequence of upstream primer is 5 '-TAGACCAGGATGGACAAGATGAT-3 ', the sequence of downstream primer is 5 '-AACCACAGGACTAAGACGCAACA-3 '.
Negative control is ultrapure water, amounts to 20 uL.
Positive control amounts to 20uL for restructuring pMD18-T-ompA plasmid.
Damping fluid is the mixing solutions of the NaCL of the Tris-HcL of 50mM and 150mM, amounts to 40uL.
The composition of PCR enzyme comprises the KCL of Tris-HcL, the 50mM of 10mM, the MgCL of 1.5mM
2And the Poymerase/Ul of 0.05U, amount to 250uL.
Lysate is by Tris-HcL50mM, EDTA2mM, NaCl100mM and account for the mixing solutions that the SDS of lysate cumulative volume 5% forms, 250uL.
Carried out following experiment in order to verify effect of the present invention:
1 materials and methods
1.1 bacterial strainMycoplasma hyopneumoniae type strain P216 is available from Chinese veterinary microorganism preservation administrative center, and 168 strains of mycoplasma hyopneumoniae living vaccine are available from Nanjing Tianbang Bio-industry Co., Ltd.; Actinobacillus pleuropneumoniae, secondary pig are had a liking for bacillus, chicken virus mycoplasma, intestinal bacteria, chicken intestinal diorder Salmonellas and are preserved by Guizhou Province's livestock and poultry pestilence research laboratory.
Main agentsGoldview, Tris, EDTA, DL2000, Taq DNA Polymerase(5U/ μ L) and corresponding 10 * Taq Buffer, dNTP etc. available from precious biological (Dalian) Engineering Co., Ltd; Phenol, chloroform, dehydrated alcohol, mycoplasma culture medium are domestic reagent.
Design of primersAccording to 1 pair of Auele Specific Primer of mycoplasma hyopneumoniae ompA gene order design, synthetic by precious biological (Dalian) Engineering Co., Ltd, upstream primer: 5 '-TAGACCAGGATGGACAAGATGAT-3 ', downstream primer: 5 '-AACCACAGGACTAAGACGCAACA-3 '.
Test kit forms
(1) 0.1moL/L PBS; (2) lysate; (3) DL2000; (4) PCR enzyme; (5) ultrapure water; (6) primer; (7) positive control; (8) negative control
1.5 the extracting of mycoplasma hyopneumoniae nucleic acid
(1) gets altogether about 50 mg of the tissue samples such as sample lung to be checked, lymphoglandula, tonsilla, add 500 μ L PBS damping fluids, 3 12000 r/min centrifuging and taking supernatants of-20 ℃ of multigelations were used for the extraction of nucleic acid after sample to be checked ground, and extracted mycoplasma hyopneumoniae DNA with phenol chloroform method.
(2) mycoplasma hyopneumoniae type strain, 168 strains of mycoplasma hyopneumoniae living vaccine, intestinal bacteria, actinobacillus pleuropneumoniae, secondary pig are had a liking for bacillus, chicken virus mycoplasma, the pure bacterium liquid of chicken intestinal diorder Salmonellas, and the nose swab sample all extracts DNA with boiling method.
The optimization of reaction conditions
To the PCR reaction conditions, comprise annealing temperature (50 ℃, 55 ℃, 60 ℃), primer concentration (5 μ mol/L, 10 μ mol/L, 20 μ mol/L), Taq DNA polymerase concentration (0.5 U, 1 U, 2 U) is optimized, to determine optimum reaction condition, simultaneously with ultrapure water as blank.10 * Taq Buffer, 5 μ L, dNTP mixture 4 μ L are carried out in the PCR reaction in 25 μ L reaction systems; Taq DNA polymerase 1 μ L complements to 25 μ L with ultrapure water.Reaction conditions is: 94 ℃ of 5 min; 94 ℃ of 1 min, annealing temperature (50 ℃, 55 ℃, 60 ℃) 1 min, 72 ℃ of 1 min, 30 circulations; Last 72 ℃ are extended 10 min.Get 7 μ L pcr amplification products and in 10 g/L sepharoses, carry out the electrophoresis evaluation.
Sensitivity test
After the mycoplasma hyopneumoniae type strain nucleic acid that extracts measured concentration with protein nucleic acid instrument, carry out carrying out respectively the PCR reaction behind 10 doubling dilutions, with the susceptibility of the PCR diagnostic kit determining to set up.
Specific test
Have a liking for bacillus, chicken virus mycoplasma, chicken intestinal diorder Salmonellas with mycoplasma hyopneumoniae type strain, 168 strains of porcine mycoplasmal pneumonia living vaccine, intestinal bacteria, actinobacillus pleuropneumoniae, secondary pig and carry out the PCR reaction, with the specificity of the PCR diagnostic kit determining to set up.
Replica test
PCR method is set up in application, 3 reliabilities with assay of duplicate detection mycoplasma hyopneumoniae DNA sample.
The preservation period of test kit detects
Test kit is kept at 4 ℃ ,-20 ℃ respectively at March, June, 1 year, positive is detected, to determine the shelf time of test kit.
Test kit is to the detection of clinical sample
58 parts of pathological material of diseases to Guiyang City, Guizhou Province, In Qiannan, Tongren Prefecture, several large scale of pig farm field, Wengan County and self-employed pig raiser's collection in 2010～2012 years detect.The extracting of DNA nucleic acid is carried out bacteriology and biochemical investigation simultaneously with reference to 1.5.
The result
2.1 the evaluation of PCR product
The pcr amplified fragment of mycoplasma hyopneumoniae reclaims through glue, send the order-checking of Dalian precious biotechnology company limited, and the result shows that amplified fragments is respectively the specific band of mycoplasma hyopneumoniae.
The optimization of reaction conditions
PCR reaction optimum reaction condition in 25 μ L reaction systems is 55 ℃ of annealing temperatures, Taq DNA polymerase1 U, primer concentration 10 μ mol/L have all effectively amplified its purpose fragment with primer, clip size is 500 bp, and specific fragment produces nothing but, as shown in Figure 1.
Sensitivity test
In the PCR reaction, the detection limit of mycoplasma hyopneumoniae DNA is 0.25 ng/L, as shown in Figure 2.
Specific test
Have a liking for bacillus, chicken virus mycoplasma, chicken intestinal diorder Salmonellas with mycoplasma hyopneumoniae type strain, 168 strains of mycoplasma hyopneumoniae living vaccine, intestinal bacteria, actinobacillus pleuropneumoniae, secondary pig and carry out the PCR reaction, specificity with definite PCR diagnostic kit of setting up, mycoplasma hyopneumoniae type strain, 168 strains of mycoplasma hyopneumoniae living vaccine are positive as a result, and intestinal bacteria, actinobacillus pleuropneumoniae, secondary pig are had a liking for bacillus, chicken virus mycoplasma, the negative (see figure 3) of chicken intestinal diorder Salmonellas.
Replica test
Use the PCR method of setting up, duplicate detection mycoplasma hyopneumoniae DNA sample 3 times, the result is all consistent.
The preservation period of test kit detects
Test kit is kept at 4 ℃ ,-20 ℃ respectively at January, March, June, 1 year, positive is detected, 1 year band of 4 ℃ of preservations is lighter, preserves January, March, June, 1 year band brightness without impact for-20 ℃.
Test kit is to the detection of clinical sample
58 parts of pathological material of diseases to Guiyang City, Guizhou Province, In Qiannan, Tongren Prefecture, several large scale of pig farm field, Wengan County and self-employed pig raiser's collection in 2010～2012 detect, and carry out simultaneously bacteriology and biochemical investigation.Detect altogether 25 minutes positive, and consistent with bacteriology and biochemical investigation result.
Discuss
The diagnostic method that is used at present mycoplasma hyopneumoniae is a lot, but mainly is that the susceptibility of PCR diagnostic kit, specificity, repeatability, accuracy have no report take mycoplasma hyopneumoniae antibody test ELISA diagnostic kit as main on the market.Mycoplasma hyopneumoniae is high to nutritional requirement, thereby the separation and Culture of mycoplasma hyopneumoniae difficulty (Cheng Xiaoying etc., 2006) relatively.Traditional diagnostic method needs to carry out Comprehensive Diagnosis in conjunction with clinical symptom and histopathology pathology, bacteria isolation and identification, just can obtain accurately and reliably diagnostic result, grow (Jia Rongli etc., 2002) consuming time.Yu Min etc. (2003) are according to a pair of Auele Specific Primer of having delivered Mhp 16SrRNA gene design abroad, and the specific fragment that to amplify a size be 653bp shows that for the sensitivity experiments of PCR this can detect the DNA of 1ng to primer.Liu Maojun etc. (2009) set up mycoplasma hyopneumoniae PCR detection method, and the method have been carried out specificity and sensitivity test according to 1 pair of primer of P36 gene design of mycoplasma hyopneumoniae, and susceptibility has reached 0.426ng.The investigator designs 1 pair of special primer and TaqMan probe according to the Mhp conservative region simultaneously, has set up fluorescent quantitative PCR detection method that can be quantitative to the Mhp culture, and sensitivity can reach 13 copy/L(Wang Jianbos etc., 2009).But the cost of the method diagnosis is higher, and what veterinary laboratories at county level mainly were equipped with at present is conventional PCR instrument, so apply at the anti-station of beast that the method is not suitable in county's one-level.The present invention has designed pair of primers according to the Actinobacillus pleuropneumoniae mycoplasma hyopneumoniae ompA gene order among the GenBank, by the PCR reaction conditions is optimized, has developed detection porcine mycoplasmal pneumonia PCR diagnostic kit.Show the advantages such as test kit of the present invention has fast, sensitive, accurate, good reproducibility, long quality-guarantee period through above-mentioned experimental result, although the diagnosis porcine mycoplasmal pneumonia is not investigator's final purpose, make rational prophylactico-therapeutic measures by diagnostic result and be only final purpose to reach the purification porcine mycoplasmal pneumonia, but utilize the present invention can be with the early clinic quick diagnosis that realizes porcine mycoplasmal pneumonia, prevention and the treatment of porcine mycoplasmal pneumonia had great importance.
Description of drawings
Accompanying drawing 1 is Mhp PCR of the present invention;
M:DL 2000; 1:Mhp PCR product (500 bp); 2: negative control;
Accompanying drawing 2 sensitivity test results of the present invention;
M:DL 2000; 1-7:10
-1～10
-7The Mhp DNA PCR result of dilution;
Accompanying drawing 3 is specific test result of the present invention;
M:DL 2000; 1: the porcine mycoplasmal pneumonia type strain; 2: 168 strains of porcine mycoplasmal pneumonia living vaccine; 3: intestinal bacteria; 4: actinobacillus pleuropneumoniae; 5: secondary pig is had a liking for bacillus; 6: chicken virus mycoplasma; 7: the chicken intestinal diorder Salmonellas.
Embodiment
Embodiments of the invention: porcine mycoplasmal pneumonia PCR diagnostic kit, it comprises that concentration is the PBS40uL of 0.1moL/L, lysate 250uL, DL2000100uL, PCR enzyme 250uL, ultrapure water 170uL, primer 40uL, positive control 20uL and negative control 20uL; Wherein primer comprises upstream primer and downstream primer, each 20 uL of upstream primer and downstream primer, and the sequence of upstream primer is 5 '-TAGACCAGGATGGACAAGATGAT-3 ', the sequence of downstream primer is 5 '-AACCACAGGACTAAGACGCAACA-3 '; Negative control is ultrapure water; Positive control is restructuring pMD18-T-ompA plasmid; Damping fluid is the mixing solutions of the NaCL of the Tris-HcL of 50mM and 150mM, amounts to 40uL; The composition of PCR enzyme comprises the KCL of Tris-HcL, the 50mM of 10mM, the MgCL of 1.5mM
2And the Poymerase/Ul of 0.05U; Lysate is by Tris-HcL50mM, EDTA2mM, NaCl100mM and account for the mixing solutions that the SDS of lysate cumulative volume 5% forms.
SEQUENCE LISTING
Sequence table
＜110〉Guizhou Farming Animal Science and Veterinary Research Institute
＜120〉a kind of porcine mycoplasmal pneumonia PCR diagnostic kit
<160> 2
<210> 1
<211> 23
<212> DNA
＜213〉artificial sequence
<220>
＜223〉according to the mycoplasma hyopneumoniae ompA gene order of logining among the GenBank, use the DNAStar software design, to be used for pcr amplification.
<400> 1
TAGAC CAGGA TGGAC AAGAT GAT 23
<210> 2
<211> 23
<212> DNA
＜213〉artificial sequence
<220>
＜223〉according to the mycoplasma hyopneumoniae ompA gene order of logining among the GenBank, use the DNAStar software design, to be used for pcr amplification.
<400> 2
AACCA CAGGA CTAAG ACGCA ACA 23
Claims (6)
1. porcine mycoplasmal pneumonia PCR diagnostic kit, it is characterized in that: it comprises that concentration is the PBS40uL of 0.1moL/L, lysate 250uL, DL2000100uL, PCR enzyme 250uL, ultrapure water 170uL, primer 40uL, positive control 20uL and negative control 20uL; Wherein primer comprises upstream primer and downstream primer, each 20 uL of upstream primer and downstream primer, and the sequence of upstream primer is 5 '-TAGACCAGGATGGACAAGATGAT-3 ', the sequence of downstream primer is 5 '-AACCACAGGACTAAGACGCAACA-3 '.
2. porcine mycoplasmal pneumonia PCR diagnostic kit according to claim 1, it is characterized in that: negative control is ultrapure water, amounts to 20 uL.
3. porcine mycoplasmal pneumonia PCR diagnostic kit according to claim 1 is characterized in that: positive control amounts to 20uL for restructuring pMD18-T-ompA plasmid.
4. porcine mycoplasmal pneumonia PCR diagnostic kit according to claim 1 is characterized in that: damping fluid is the mixing solutions of the NaCL of the Tris-HcL of 50mM and 150mM, amounts to 40uL.
5. porcine mycoplasmal pneumonia PCR diagnostic kit according to claim 1, it is characterized in that: the composition of PCR enzyme comprises the KCL of the Tris-HcL of 10mM, 50mM, the MgCL of 1.5mM
2And the Poymerase/Ul of 0.05U, amount to 250uL.
6. porcine mycoplasmal pneumonia PCR diagnostic kit according to claim 1 is characterized in that: lysate is by Tris-HcL50mM, EDTA2mM, NaCl100mM and account for the mixing solutions that the SDS of lysate cumulative volume 5% forms, 250uL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310021475.4A CN103014174B (en) | 2013-01-22 | 2013-01-22 | MPS (Macoplasmal Pneumoniae of Swine) PCR (Polymerase Chain Reaction) diagnostic kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310021475.4A CN103014174B (en) | 2013-01-22 | 2013-01-22 | MPS (Macoplasmal Pneumoniae of Swine) PCR (Polymerase Chain Reaction) diagnostic kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103014174A true CN103014174A (en) | 2013-04-03 |
| CN103014174B CN103014174B (en) | 2015-01-14 |
Family
ID=47963286
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310021475.4A Expired - Fee Related CN103014174B (en) | 2013-01-22 | 2013-01-22 | MPS (Macoplasmal Pneumoniae of Swine) PCR (Polymerase Chain Reaction) diagnostic kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103014174B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105950719A (en) * | 2016-04-28 | 2016-09-21 | 上海源培生物科技股份有限公司 | Mycoplasma detecting and removing method |
| CN107058506A (en) * | 2017-02-20 | 2017-08-18 | 河南省农业科学院畜牧兽医研究所 | A kind of kit of Direct PCR detection porcine mycoplasmal pneumonia cause of disease and its application |
| CN109182603A (en) * | 2018-09-30 | 2019-01-11 | 广西壮族自治区兽医研究所 | A kind of the multiple PCR detection primer group and kit of quick differentiation PCV2, PCV3 and Mhp |
| CN109234418A (en) * | 2018-11-20 | 2019-01-18 | 湖南新南方养殖服务有限公司 | A kind of primer, kit and method identifying mycoplasma hyopneumoniae street strain and vaccine strain |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103571960A (en) * | 2013-11-08 | 2014-02-12 | 广东温氏食品集团股份有限公司 | Absolute fluorescent quantitative polymerase chain reaction (PCR) primer pair, probe and method for determining growth titer of mycoplasma hyopneumoniae |
-
2013
- 2013-01-22 CN CN201310021475.4A patent/CN103014174B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103571960A (en) * | 2013-11-08 | 2014-02-12 | 广东温氏食品集团股份有限公司 | Absolute fluorescent quantitative polymerase chain reaction (PCR) primer pair, probe and method for determining growth titer of mycoplasma hyopneumoniae |
Non-Patent Citations (1)
| Title |
|---|
| WILTON,J. ET AL.: "AF540381.1", 《NCBI GENBANK》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105950719A (en) * | 2016-04-28 | 2016-09-21 | 上海源培生物科技股份有限公司 | Mycoplasma detecting and removing method |
| CN107058506A (en) * | 2017-02-20 | 2017-08-18 | 河南省农业科学院畜牧兽医研究所 | A kind of kit of Direct PCR detection porcine mycoplasmal pneumonia cause of disease and its application |
| CN109182603A (en) * | 2018-09-30 | 2019-01-11 | 广西壮族自治区兽医研究所 | A kind of the multiple PCR detection primer group and kit of quick differentiation PCV2, PCV3 and Mhp |
| CN109234418A (en) * | 2018-11-20 | 2019-01-18 | 湖南新南方养殖服务有限公司 | A kind of primer, kit and method identifying mycoplasma hyopneumoniae street strain and vaccine strain |
| CN109234418B (en) * | 2018-11-20 | 2021-08-13 | 湖南中净生物科技有限公司 | Primer, kit and method for identifying mycoplasma hyopneumoniae wild strain and vaccine strain |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103014174B (en) | 2015-01-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Suchodolski et al. | Application of molecular fingerprinting for qualitative assessment of small-intestinal bacterial diversity in dogs | |
| Pal et al. | Rapid detection and differentiation of Erysipelothrix spp. by a novel multiplex real‐time PCR assay | |
| CN102363815B (en) | Reagent for detecting salmonellae by using cross primer nucleic acid isothermal amplification technology, amplification method and detection method for salmonellae | |
| Foley et al. | Comparative analyses of the quality and yield of genomic DNA from invasive and noninvasive, automated and manual extraction methods | |
| CN104059977A (en) | Salmonella serotype identification method and kit thereof | |
| CN103014174B (en) | MPS (Macoplasmal Pneumoniae of Swine) PCR (Polymerase Chain Reaction) diagnostic kit | |
| CN101481742B (en) | Detection kit for Mycoplasma hyopneumoniae and use thereof | |
| Fernandes et al. | Brucella canis infection in dogs attended in veterinary clinics from Patos, Paraíba state, Brazil | |
| Figueiredo et al. | Detection of Mycobacterium bovis DNA in nasal swabs from tuberculous cattle by a multiplex PCR | |
| CN103305613B (en) | Giant salamander pathogenic hydrophila gingivalis PCR diagnostic kit | |
| CN103397023B (en) | Bacillus erysipelatos-suis PCR (Polymerase Chain Reaction) primer and application thereof | |
| Abtin et al. | Isolation and identification of Mycoplasma agalactiae by culture and polymerase chain reaction (PCR) from sheep of Qom province, Iran | |
| CN102936621B (en) | Bacillus cereus detection method and kit | |
| WO2015018156A1 (en) | Detection kit and detection method for bacteria of vibrio genus | |
| CN103993072B (en) | The multiple PCR detection kit of the pathogenic sweetfish pseudomonas of a kind of Rapid identification and method thereof | |
| Wani et al. | Isolation and characterization of Dichelobacter nodosus from ovine and caprine footrot in Kashmir, India | |
| Nayel et al. | Molecular typing of Clostridium perfringens isolates from soil, healthy, and diseased sheep in Egypt by multiplex PCR | |
| CN106520986A (en) | Quintuple PCR (polymerase chain reaction) detection method capable of detecting multiple pathogens simultaneously | |
| CN102146468B (en) | Special primer for assisted identification of Streptococcus suis type 2 and Streptococcus suis type 7 and application thereof | |
| CN106319080A (en) | PCR detection kit for rapidly identifying mycoplasma hyopeumoniae, mycoplasma synoviae and mycoplasma hyorhinis, and applications of PCR detection kit | |
| Rahimabadi et al. | Isolation and identification of Mycoplasma agalactiae by culture and polymerase chain reaction methods in the sheep herds in Guilan province, Iran. | |
| Jiang et al. | Rapid on-the-spot detection of Edwardsiella piscicida using recombinase polymerase amplification with lateral flow | |
| CN103122383B (en) | Horizontal fluorescent quantitation polymerase chain reaction (PCR) detection method of streptococcus suis strain | |
| CN104032000A (en) | Method and kit for detecting bacillus cereus | |
| CN104894232A (en) | Citrobacter freundii fluorescent quantitative PCR diagnostic reagent kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150114 Termination date: 20190122 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |